Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Here, we report a newly designed, affinity pulldown platform using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA for rapid and cost-effective analyses of plasma LMW biothiols. In the presence of organic hydroperoxide, LMW biothiols triggered the rapid dissociation of FAM-labeled dsDNA from FLAG-tagged OhrRBS via S-thiolation of OhrRBS on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pulldown. This method was easily extended by using a reducing agent to detect free and total LMW biothiols simultaneously in mouse plasma. Unlike free plasma LMW biothiols, total plasma LMW biothiols were more elevated in ΔLDLR mice than those in normal mice. Owing to the rapid dissociation of OhrR/dsDNA complexes in response to LMW biothiols, this pulldown platform is immediately suitable for monitoring rapid redox changes in plasma LMW biothiols as well as studying oxidative stress and diseases in blood.